CRISPR-Cas9 mediated gene editing to enhance cover crops

Abstract

Cover crops provide environmental benefits and a potential source of sustainable oil, but their uncertain economic viability remains a challenge. Specialty oils, valuable even at lower production volumes, are often derived from species with poor agronomic traits. Camelina sativa L. Crantz (camelina) and Thlaspi arvense L. (pennycress) are oilseed cover crops amenable to metabolic engineering and genetic transformation. Increasing the value of the oil produced in these crops through metabolic engineering addresses a key barrier to implementation and could encourage higher cover-cropping rates among farmers. Efficiently redirecting carbon flux towards engineered triacylglycerols (TAGs) requires minimizing competition from endogenous TAG biosynthesis pathways. We hypothesize that disrupting native Diacylglycerol O-acyltransferase 1 (DGAT1) and Phospholipid:diacylglycerol acyltransferase 1 (PDAT1), the terminal enzymes in TAG biosynthesis, would enhance the accumulation of specialty TAGs produced by transgenic acyltransferases. Eliminating endogenous DGAT1 and PDAT1 activity effectively channels diacylglycerol (DAG) towards transgenic TAG-synthesizing enzymes, boosting specialty TAG production. Previous work demonstrated the potential of RNAi-mediated DGAT1 knockdown to increase specialty-TAG production (Alkotami et al. 2024). However, RNAi-based gene silencing can exhibit variable knockdown efficiency and residual enzyme activity remains. Using CRISPR-Cas9, we generated dgat1 and pdat1 knockout lines in both camelina and pennycress. CRISPR-Cas9 knockouts offer a genetically stable solution for long-term metabolic engineering by eliminating enzyme activity. These results demonstrate a scalable and stable genetic engineering strategy to enhance the commercial potential of cover crops by tailoring seed oil composition for diverse industrial applications.